Hepatitis C virus (HCV) infection is a major health problem in both developed and developing countries. It is estimated that about 1 to 5% of the world population is affected by the virus. HCV infection appears to be the most important cause of transfusion-associated hepatitis and frequently progresses to chronic liver damage. Moreover, evidence exists implicating HCV in induction of hepatocellular carcinoma. Consequently, the demand for reliable diagnostic methods and effective therapeutic agents is high. Also sensitive and specific screening methods of HCV-contaminated blood-products and improved methods to culture HCV are needed.
HCV is a positive stranded RNA virus of approximately 9,600 bases which encode a single polyprotein precursor of about 3000 amino acids. Proteolytic cleavage of the precursor coupled to co- and posttranslational modifications has been shown to result in at least three structural and six non-structural proteins. Based on sequence homology, the structural proteins have been functionally assigned as one single core protein and two envelope glycoproteins: E1 and E2. The E1 protein consists of 192 amino acids and contains 4 to 5 N-glycosylation sites, depending on the HCV genotype. The E2 protein consists of 363 to 370 amino acids and contains 9 to 11 N-glycosylation sites, depending on the HCV genotype (for reviews see: Major and Feinstone, 1997; Maertens and Stuyver, 1997). The E1 protein contains various variable domains (Maertens and Stuyver, 1997). The E2 protein contains three hypervariable domains, of which the major domain is located at the N-terminus of the protein (Maertens and Stuyver, 1997). The HCV glycoproteins localize predominantly in the ER where they are modified and assembled into oligomeric complexes.
In eukaryotes, sugar residues are commonly linked to four different amino acid residues. These amino acid residues are classified as O-linked (serine, threonine, and hydroxylysine) and N-linked (asparagine). The O-linked sugars are synthesized in the Golgi or rough Endoplasmic Reticulum (ER) from nucleotide sugars. The N-linked sugars are synthesized from a common precursor, and subsequently processed. It is believed that HCV envelope proteins are N-glycosylated. It is known in the art that addition of N-linked carbohydrate chains is important for stabilization of folding intermediates and thus for efficient folding, prevention of malfolding and degradation in the endoplasmic reticulum, oligomerization, biological activity, and transport of glycoproteins (see reviews by Rose et al., 1988; Doms et al., 1993; Helenius, 1994). The tripeptide sequences Asn-X-Ser and Asn-X-Thr (in which X can be any amino acid) on polypeptides are the consensus sites for binding N-linked oligosaccharides. After addition of the N-linked oligosaccharide to the polypeptide, the oligosaccharide is further processed into the complex type (containing N-acetylglucosamine, mannose, fucose, galactose and sialic acid) or the high-mannose type (containing N-acetylglucosamine and mannose). HCV envelope proteins are believed to be of the high-mannose type. N-linked oligosaccharide processing in yeast is very different from mammalian Golgi processing. In yeast the oligosaccharide chains are elongated in the Golgi through stepwise addition of mannose, leading to elaborate high mannose structures, referred to as hyperglycosylation. In contrast therewith, proteins expressed in prokaryotes are never glycosylated.
Patterns of high mannose-type glycosylation of proteins or peptides have been determined for a variety of eukaryotic cells. In mammalian cells, an average of 5 to 9 mannose units is linked to two N-acetylglucosamine moieties in a core-glycosylation-type oligosaccharide (the structure represented in short as Man(5-9)GlcNAc(2)). Core-glycosylation refers to a structure similar to the boxed structure in FIG. 3 of Herscovics and Orleans (1993).
The methylotrophic yeast Pichia pastoris was reported to attach an average of 8 to 14 mannose units, i.e. Man(8-14)GlcNAc(2) per glycosylation site (Tschopp in EP0256421) and approximately 85% of the N-linked oligosaccharides are in the size range Man(8-14)GlcNAc(2) (Grinna and Tschopp 1989). Other researchers have published slightly different oligosaccharide structures attached to heterologous proteins expressed in P. pastoris: Man(8-9)GlcNAc(2) (Montesino et al. 1998), Man(9-14)GlcNAc(2) or Man(9-15)GlcNAc(2) (Kalidas et al. 2001), and Man(8-18)GlcNAc(2) with a preponderence of Man(9-12)GlcNAc(2) and with the major overall oligosaccharide being Man(10)GlcNAc(2) (Miele et al. 1998). Trimble et al. (1991) reported an equal distribution of Man(8)GlcNAc(2) and Man(9)GlcNAc(2) in about 75% of the N-linked oligosaccharides with additionally 17% of the N-glycosylation sites being occupied by Man(10)GlcNAc(2) and the remaining 8% of the sites by Man(11)GlcNAc(2). Hyperglycosylation of a P. pastoris-expressed protein has been reported occasionally (Scorer et al. 1993).
Aspergillus niger is adding Man(5-10)GlcNAc(2) to N-glycosylation sites (Panchal and Wodzinski 1998).
The Saccharomyces cerevisiae glycosylation deficient mutant mnn9 differs from wild-type S. cerevisiae in that mnn9 cells produce glycosylated proteins with a modified oligosaccharide consisting of Man(9-13)GlcNAc(2) instead of hyperglycosylated proteins (Mackay et al. in U.S. Pat. No. 5,135,854 and Kniskern et al. in WO94/01132). Another S. cerevisiae mutant, och1mnn9, was reported to add Man(8)GlcNAc(2) to N-glycosylation sites in proteins (Yoshifumi et al. JP06277086).
Characteristic for S. cerevisiae (wild-type and mnn9 mutant) core oligosaccharides is the presence of terminal α1,3-linked mannose residues (Montesino et al. 1998). Oligosaccharides attached to N-glycosylation sites of proteins expressed in P. pastoris or S. cerevisiae och1mnn1 are devoid of such terminal α1,3-linked mannoses (Gellissen et al. 2000). Terminal α1,3-linked mannoses are considered to be allergenic (Jenkins et al. 1996). Therefor, proteins carrying on their oligosaccharides terminal α1,3-linked mannose residues are not suitable for diagnostic or therapeutic purposes.
The glycosylation pattern on proteins expressed in the methylotrophic yeast Hansenula polymorpha have, despite the use of this yeast for production of a considerable number of heterologous proteins (see Table 3 in Gellissen et al. 2000), not been studied in great detail. From the experiments of Janowicz et al. (1991) and Diminsky et al. (1997), it seems that H. polymorpha is not or only poorly glycosylating the large or small hepatitis B viral surface antigen (HBsAg). Most likely this is due to the fact that the HBsAg was expressed without signal peptide, thus preventing the produced HBsAg to enter the lumen of the endoplasmic reticulum (ER) and glycosylation. Limited addition of mono- or dihexoses to G-CSF (granulocyte colony stimulating factor) produced in H. polymorpha was reported (Fischer et al. in WO00/40727). At the other hand, hyperglycosylation was observed of a heterologous α-galactosidase expressed in H. polymorpha cells (Fellinger et al. 1991).
To date, vaccination against disease has been proven to be the most cost effective and efficient method for controlling diseases. Despite promising results, efforts to develop an efficacious HCV vaccine, however, have been plagued with difficulties. A condition sine qua non for vaccines is the induction of an immune response in patients. Consequently, HCV antigenic determinants should be identified, and administered to patients in a proper setting. Antigenic determinants can be divided in at least two forms, i.e. lineair and conformational epitopes. Conformational epitopes result from the folding of a molecule in a three-dimensional space, including co- and posttranslational modifications, such as glycosylation. In general, it is believed that conformational epitopes will realize the most efficacious vaccines, since they represent epitopes which resemble native-like HCV epitopes, and which may be better conserved than the actual linear amino acid sequence. Hence, the eventual degree of glycosylation of the HCV envelope proteins is of the utmost importance for generating native-like HCV antigenic determinants. However, there are seemingly insurmountable problems with culturing HCV, that result in only minute amounts of virions. In addition, there are vast problems with the expression and purification of recombinant proteins, that result in either low amounts of proteins, hyperglycosylated proteins, or proteins that are not glycosylated.
The HCV envelope proteins have been produced by recombinant techniques in Escherichia coli, insect cells, yeast cells and mammalian cells. However, expression in higher eukaryotes has been characterised by the difficulty of obtaining large amounts of antigens for eventual vaccine production. Expression in prokaryotes, such as E. coli results in HCV envelope proteins that are not glycosylated. Expression of HCV envelope proteins in yeast resulted in hyperglycosylation. As already demonstrated by Maertens et al. in WO 96/04385, the expression of HCV envelope protein E2 in Saccharomyces cerevisiae leads to proteins which are heavily glycosylated. This hyperglycosylation leads to shielding of protein epitopes. Although Mustilli et al. (1999) claims that expression of HCV E2 in S. cerevisiae results in core-glycosylation, the results of the intracellularly expressed material demonstrate that part of it is at least hyperglycosylated, while the correct processing of the remainder of this material has not been shown. Moreover, the hyperglycosylation observed by Mustilli et al. (1999) could only be prevented in the presence of tunicamycin, an inhibitor of glycosylation, and this does thus not reflect glycosylation occurring under normal, natural growth conditions. The need for HCV envelope proteins derived from an intracellular source is well accepted (Maertens et al. in WO 96/04385, Heile et al., 2000). This need is further exemplified by the poor reactivity of the secreted yeast derived E2 with sera of chimpanzee immunized with mammalian cell culture derived E2 proteins as evidenced in FIG. 5 of Mustilli et al (1999). This is further documented by Rosa et al (1996) who show that immunization with yeast derived HCV envelope proteins fails to protect from challenge.
Consequently, there is a need for efficient expression systems resulting in large and cost-effective amounts of proteins that at the same time have a native-like glycosylation pattern devoid of terminal α1,3-linked mannoses. In particular, such systems are needed for production of HCV envelope proteins.